Advanced Research Laboratory, Hitachi Ltd., Hatoyama, Saitama, Japan.
Biotechnol Bioeng. 1991 Jun 5;38(1):37-42. doi: 10.1002/bit.260380106.
Fed-batch cultures of recombinant E. coli HB101 harboring expression plasmid pTRLBT1 or pTREBT1, with acetate concentration monitoring, are investigated to obtain high cell density and large amounts of human epidermal growth factor (hEGF). The expression plasmid pTRlBT1 contains a synthetic hEGF gene attached downstream of the N-terminal fragment of the trp L gene preceded by the trp promoter. The expression plasmid pTREBT1 contains the same coding sequence attached downstream of the N-terminal fragment of the trp E gene preceded by the trp promoter, trp L gene, and attenuator region. E. coli harboring pTREBT1 produces 0.56 mg/L hEGE and immediately degrades it. On the other hand E. coli harboring pTRLBT1 produces 6.8 mg/L hEGF and does not decompose it. Prominent inclusion bodies are observed in E. coli cells harboring pTRLBT1 using an election microscope. To Cultivate E. coli harboring pTRLBT1, a fed-batch culture system, divided into a cell growth step and an hEGF production step, is carried out. The cells grow smoothly without acetate-induced inhibition. Cell concentration and hEGF quantity reach the high values of 21 g/L and 60 mg/L, respectively.
含表达质粒 pTRLBT1 或 pTREBT1 的重组大肠杆菌 HB101 的分批补料培养，同时监测乙酸浓度，以获得高细胞密度和大量的人表皮生长因子（hEGF）。表达质粒 pTRlBT1 含有一个合成的 hEGF 基因，该基因附着在色氨酸操纵子的 trp L 基因的 N 端片段下游，前面是 trp 启动子。表达质粒 pTREBT1 含有相同的编码序列，附着在色氨酸操纵子的 trp E 基因的 N 端片段下游，前面是 trp 启动子、trp L 基因和衰减子区域。携带 pTREBT1 的大肠杆菌产生 0.56mg/L 的 hEGE 并立即将其降解。另一方面，携带 pTRLBT1 的大肠杆菌产生 6.8mg/L 的 hEGF 且不会分解它。使用电子显微镜观察到携带 pTRLBT1 的大肠杆菌细胞中存在明显的包涵体。为了培养携带 pTRLBT1 的大肠杆菌，采用分批补料培养系统，分为细胞生长阶段和 hEGF 生产阶段。细胞生长平稳，没有乙酸诱导的抑制。细胞浓度和 hEGF 量分别达到 21g/L 和 60mg/L 的高值。
